Download Newly identified cross-membranes orchestrate

Newly identified cross-membranes orchestrate compartmentalization in substrate
hyphae of Streptomycetes
Joost Willemse1*, K. Celler1, R. I. Koning2, A.J. Koster2 , Gilles P. van Wezel1
1
Department of Molecular Biotechnology, IBL, Leiden University, PO Box 9505, 2300
AB Leiden, The Netherlands
2
Department of MCB, LUMC, PO Box 9600, 2300 RC Leiden, The Netherlands.
[email protected]
Key words: cryo-electron tomography, cell division, FRAP, Streptomyces
Introduction. Streptomyces are filamentous soil-dwelling saprophytes, well-known for their
complex multicellular life style and their ability to produce a plethora of natural products,
including antibiotics, anticancer agents and immunosuppressants. After spore germination,
hyphae grow by tip extension and branching to form a syncytial vegetative mycelium,
occasionally separated by peptidoglycan-based septa or cross-walls. Cell division is not
essential for growth in Streptomyces as shown by the fact that the gene for the cell-division
scaffold protein FtsZ can be deleted. Surprisingly, the ftsZ-deletion mutant can be propagated
from fragments obtained by mechanical maceration of mycelia, which suggests the presence
of additional compartmentalization of the hyphae, besides that by peptidoglycan-based crosswalls. We have now demonstrated the presence of a membrane system in the substrate hyphae
of Streptomyces, whereby so-called cross-membranes form protein-impermeable barriers,
independent of cross-walls or the cell division machinery. Similar cross-membranes were
discovered in very young vegetative hyphae.
Results. Membrane phospholipids were studied
with fluorescence light microscopy. This revealed
that 75% of membrane structures are independent
of peptidoglycan cross-walls. These assemblies
can be up to several micrometres in length, are
capable of spanning the width of the vegetative
hyphae. We then applied cryo-correlative light
and electron microscopy, combining cryo-light
microscopy with cryo-electron tomography, to
study the fine structural detail of the membrane
assemblies. These assemblies within hyphae
revealed the formation of bundles of tube-like
structures varying from small assemblies along
the cell wall to large structures completely delimiting the hyphae. Tomograms show that the
membrane assemblies form in the space between the cytoplasmic membrane and cell wall.
Large hyphae-spanning membrane assemblies were devoid of DNA, and served as a starting
point for septum synthesis (Figure, middle and bottom images). To confirm that membranes
formed independent of septa, we also studied the S. coelicolor ftsZ deletion strain, which
grows without forming septa. Membrane formations in this strain were indistinguishable and
as frequent as wild type membrane formation.
To assess if the large cross-membrane structures can form cytosol-impermeable barriers, we
applied fluorescence recovery after photobleaching (FRAP), whereby GFP molecules were
bleached in mycelial compartments, to see if GFP could cross the membrane barriers to
restore fluorescence. These experiments demonstrated that some 30% of the cross-membranes
are cytosol-impermeable. Importantly, the cross-membranes form DNA-free zones in the
hyphae, offering a solution to the long-standing problem of how cell-wall remodelling and
cell division can take place without DNA damage in the multinucleoid hyphae.
References.
1. Celler K, Koning R., Willemse J, Koster A, van Wezel G. (2016). Nature Communications.
7:1-8.
2. Yagüe P, Willemse J, Koning R., Rioseras B, Lopez-Garcia M, Gonzalez-Quiñonez N,
Iglesias C, Shliaha P, Rogowska-Wrzesinska A, Koster A, Jensen O, van Wezel G, Manteca A.
(2016), Nature Communications. :12467.